Ntrk2 fusions

ABSTRACT

The invention provides to NTRK (“Neurotrophic Tyrosine Receptor Kinase) gene fusions, NTRK. fosion proteins, and fragments of those genes and polypeptides. The invention further provides methods of diagnosing and treating diseases or disorders associated with NTRK fusions, such as conditions mediated by aberrant NTRK expression or activity, or overexpression of

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/860,153, filed Jul. 30, 2013, and U.S. Provisional Application No.61/943,028, filed Feb. 21, 2014, the contents of both of which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to NTRK2 (Neurotrophic Tyrosine Receptor Kinase)gene fusions and NTRK2 fusion proteins. The invention further relates tomethods of diagnosing and treating diseases or disorders associated withNTRK2 fusions, such as conditions mediated by aberrant NTRK2 expressionor activity, or conditions associated with overexpression of NTRK2.

BACKGROUND

Many forms of cancer are caused by genetic lesions that give rise totumor initiation and growth. Genetic lesions may include chromosomalaberrations, such as translocations, inversions, deletions, copy numberchanges, gene expression level changes, and somatic and germlinemutations. Indeed, the presence of such genomic aberrations is ahallmark feature of many cancers, including, for example, B cell cancer,lung cancer, breast cancer, ovarian cancer, pancreatic cancer, and coloncancer. In some models, cancer represents the phenotypic end-point ofmultiple genetic lesions that endow cells with a full range ofbiological properties required for tumorigenesis.

Recent efforts by The Cancer Genome Atlas (TCGA), the InternationalCancer Genome Consortium (ICGC), and dozens of other large-scaleprofiling efforts have generated an enormous amount of new sequencingdata for dozens of cancer types this includes whole-genome DNA,whole-exome DNA, and full-transcriptome RNA sequencing. These effortshave led to the identification of new driver genes and fusion geneswithin multiple cancer types. Fusions, particularly fusions involvingkinases, are of particular interest, as such fusions have been shown tobe oncogenic, and have been successfully targeted by new therapeutics.For example, anaplastic lymphoma kinase (ALK), one of the receptortyrosine kinases, is known to become oncogenic when fused with variousgenes. See, e.g., M. Soda et al, “Identification of the transformingEML4-ALK fusion gene in non-small-cell lung cancer,” Nature 444:561-566(2007).

A need exists for identifying novel genetic lesions associated withcancer. For example, the presence of fusions involving a kinase insamples collected from more than one source can indicate that the kinaseis an oncogenic driver. The identification of such fusions can be aneffective approach to diagnosis of cancers and development of compounds,compositions, methods, and assays for evaluating and treating cancerpatients.

SUMMARY

In one aspect, the invention provides methods for detecting the presenceof an NTRK2 fusion in a biological sample. The methods include the stepsof: (a) obtaining a biological sample from a mammal; and (b) contactingthe sample with a reagent that detects an NTRK2 fusion, to determinewhether an NTRK2 fusion is present in the biological sample. In someembodiments, the sample can be from, e.g., a cancer patient, such as,e.g., a lung cancer patient, a glioma patient, or a squamous cellcarcinoma patient. In some embodiments, the fusion can be, e.g., aTRIM24:NTRK2 fusion, an AFAP1:NTRK2 fusion, or a PAN3:NTRK2 fusion. Insome embodiments, the TRIM24:NTRK2 fusion has all or a part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:1 and SEQ ID NO:3, respectively. Insome embodiments, the TRIM24:NTRK2 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively. Insome embodiments, the AFAP1:NTRK2 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. Insome embodiments, the PAN3:NTRK2 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively.

In another aspect, the invention provides methods of diagnosing apatient having a disease or disorder associated with aberrant NTRK2expression or activity, or overexpression of NTRK2; the methods include:(a) obtaining a biological sample from the patient; and (b) contactingthe sample with a reagent that detects an NTRK2 fusion to determinewhether an NTRK2 fusion is present in the biological sample, wherein thedetection of the NTRK2 fusion indicates the presence of a disorderassociated with aberrant NTRK2 expression or activity, or overexpressionof NTRK2.

The invention also includes methods of determining a therapeutic regimenfor treating a cancer in a human subject; methods of identifying apatient likely to respond to treatment with an NTRK2 inhibitor or anNTRK2 fusion inhibitor; methods of stratifying a patient population bydetecting an NTRK2 fusion; methods of inhibiting the proliferation ofcells containing an NTRK2 fusion; methods of reducing an activity of anNTRK2 fusion; methods of treating a condition mediated by aberrant NTRK2expression or activity; methods of treating a condition characterized byoverexpression of NTRK2; methods of identifying an agent that modulatesthe activity of an NTRK2 fusion; and methods of monitoring diseaseburden in a patient having a condition mediated by NTRK2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequence of a TRIM24:NTRK2 gene fusion(SEQ ID NO:1) comprising a portion of the TRIM24 gene (NM_015905) up toand including exon 12 (amino acid number 671) and a portion of the NTRK2gene (NM_006180) starting at exon 16 (amino acid number 483). Theunderlined codons at nucleotides 2011-2013 and 2017-2019 encode the lastamino acid of TRIM24 and the first amino acid of NTRK2, respectively.The slash after nucleotide 2014 indicates the breakpoint (fusionjunction) where translocation and in-frame fusion has occurred. Theshading at nucleotides 2014-2016 indicates that nucleotides from bothTRIM24 and NTRK2 are fused in frame to form a codon and encode an aminoacid.

FIG. 2 depicts the nucleotide sequence of a TRIM24:NTRK2 gene fusion(SEQ ID NO:2) comprising a portion of the TRIM24 gene (NM_015905) up toand including exon 12 (amino acid number 671) and a portion of the NTRK2gene (NM_006180) starting at exon 15 (amino acid number 467). Theunderlined codons at nucleotides 2001-2003 and 2007-2009 encode the lastamino acid of TRIM24 and the first amino acid of NTRK2, respectively.The slash after nucleotide 2004 indicates the breakpoint wheretranslocation and in-frame fusion has occurred. The shading atnucleotides 2004-2006 indicates that nucleotides from both TRIM24 andNTRK2 are fused in frame to form a codon and encode an amino acid.

FIG. 3 depicts the amino acid sequence of an TRIM24:NTRK2 fusion protein(SEQ ID NO:3). The shaded amino acid at position 672 corresponds tonucleotides 2014-2016 in SEQ ID NO:1. This amino acid is encoded bynucleotides from both TRIM24 and NTRK2.

FIG. 4 depicts the amino acid sequence of an TRIM24:NTRK2 fusion protein(SEQ ID NO:4). The shaded amino acid at position 672 corresponds tonucleotides 2004-2006 in SEQ ID NO:2. This amino acid is encoded bynucleotides from both TRIM24 and NTRK2.

FIG. 5 depicts the nucleotide sequence of an AFAP1:NTRK2 gene fusion(SEQ ID NO:5) comprising a portion of the AFAP1 gene (NM_198595) up toand including exon 13 (amino acid number 548) and a portion of the NTRK2gene (NM_006180) starting at exon 12 (amino acid number 388). Theunderlined codons at nucleotides 1642-1644 and 1648-1650 encode the lastamino acid of AFAP1 and the first amino acid of NTRK2, respectively. Theslash after nucleotide 1645 indicates the breakpoint where translocationand in-frame fusion has occurred. The shading at nucleotides 1645-1647indicates that nucleotides from both AFAP1 and NTRK2 are fused in frameto form a codon and encode an amino acid.

FIG. 6 depicts the amino acid sequence of an AFAP1:NTRK2 fusion protein(SEQ ID NO:6). The shaded amino acid at position 549 corresponds tonucleotides 1645-1647 of SEQ ID NO:5. This amino acid is encoded bynucleotides from both AFAP1 and NTRK2.

FIG. 7 depicts the nucleotide sequence of a PAN3:NTRK2 gene fusion (SEQID NO:7) comprising a portion of the PAN3 gene (NM_175854) up to exonnumber 1 (amino acid 143) and a portion of the NTRK2 gene (NM_006180)from exon number 17 (amino acid 546). The underlined codons atnucleotides 427-429 and 433-435 encode the last amino acid of PAN3 andthe first amino acid of NTRK2, respectively. The slash after nucleotide430 and 431 indicates the breakpoint where translocation and in-framefusion has occurred. The shading at nucleotides 430-432 indicates thatnucleotides from both PAN3 and NTRK2 are fused in frame to form a codonand encode an amino acid.

FIG. 8 depicts the amino acid sequence of a PAN3:NTRK2 fusion protein(SEQ ID NO:8). The shaded amino acid at position 144 corresponds tonucleotides 430-432 of SEQ ID NO:7. This amino acid is encoded bynucleotides from both PAN3 and NTRK2.

EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention is based, at least in part, on the discovery of novelrecombination or translocation events in cancer patients that result inat least a fragment of an NTRK2 gene linked to a non-homologous promotervia a recombination or translocation event that may result in aberrantexpression (e.g., in a location where the kinase is not typicallyexpressed) or overexpression of the kinase domain of the NTRK2 gene andthus, an increase in kinase activity. Thus, a new patient population isidentified, which is characterized by the presence of an NTRK2 fusion,e.g., an NTRK2 gene fusion or fusion protein. This new patientpopulation suffers from or is susceptible to disorders mediated byaberrant NTRK2 expression or activity, or overexpression of NTRK2, suchas, e.g., a cancer. In another aspect of the invention, a new subtype ofcancer is identified, which is characterized by the presence of theNTRK2 fusions described herein. In some embodiments, the new patientpopulation suffers from or is susceptible to a lung cancer, glioma, orsquamous cell carcinoma characterized by the presence of an NTRK2fusion. New methods of diagnosing and treating the patient populationand the NTRK2 fusion cancer subtype are also provided.

The term “NTRK2 fusion” is used generically herein, and includes anyfusion molecule (e.g., gene, gene product (e.g., cDNA, mRNA, orprotein), and variants thereof) that includes a fragment of NTRK2 (inthe case of a nucleotide sequence, typically containing the codingregion for the kinase domain of NTRK2), and a non-homologous fragment(in the case of a nucleotide sequence, the promoter and/or the codingregion of a non-homologous gene, such that the coding sequence for thekinase domain of NTRK2 is under control of the promoter of thenon-homologous gene). An NTRK2 fusion protein generally includes thekinase domain of NTRK2. In some embodiments, the NTRK2 fusion is aTRIM24:NTRK2 fusion. In other embodiments, the NTRK2 fusion is anAFAP1:NTRK2 fusion. In yet other embodiments, the NTRK2 fusion is aPAN3:NTRK2 fusion.

NTRK2 Gene Fusions and Fusion Proteins

NTRK2 gene fusions are generated by a fusion between at least a part ofthe NTRK gene and a part of another gene as a result of a translocation(including inversion) within a chromosome or between chromosomes. As aresult of a translocation, the NTRK2 gene may be placed under thetranscriptional control of the partner gene promoter, resulting inaberrant NTRK2 expression or activity, or overexpression of NTRK2.Alternatively or additionally, the partner gene can include adimerization domain that causes NTRK2 to become constitutivelyactivated. In some exemplary embodiments, the fusion partner is TRIM24(Tripartite Motif Containing 24). In other exemplary embodiments, thefusion partner is AFAP1 (Actin Filament-Associated Protein 1). In yetother embodiments, the fusion partner is PAN3 (PABP-Dependent Poly(A)Nuclease 3).

As used herein, the 5′-region is upstream of, and the 3′-region isdownstream of; a fusion junction or breakpoint in one of the componentgenes, NTRK2 and the gene or protein that it is fused to may be referredto as “fusion partners.” Alternatively, they may be identified as an“NTRK2 gene fusion” or an “NTRK2 fusion protein,” which are collectivelytermed “NTRK2 fusions.” The NTRK2 fusions disclosed herein have a kinaseactivity. The phrase “having a kinase activity” as used in thisapplication means having an activity as an enzyme phosphorylating theside chain of an amino acid, such as tyrosine. In some embodiments, theNTRK2 fusion may include an in-frame fusion of the coding sequences ofNTRK2 and the fusion partner that introduces amino acids into the fusionprotein that are not part of NTRK2 or the fusion partner.

Reference to “all or a portion” or “all or part” of an NTRK2 gene fusionor SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:7, means that thenucleotide sequence comprises the entire NTRK2 gene fusion nucleotidesequence or a fragment of that sequence that comprises the fusionjunction breakpoint point between NTRK2 and its fusion partner (such as,e.g., TRIM24, AFAP1, or PAN3). The fragment may comprise 7, 8, 9, 10,12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, or morenucleotides spanning the fusion junction of the NTRK2 gene fusion.Reference to “all or a portion” or “all or part” of a NTRK2 fusionprotein or SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, meansan amino acid sequence that comprises the entire NTRK2 fusion proteinamino acid sequence or a fragment of that sequence that comprises thefusion junction breakpoint point between NTRK2 and its fusion partner(such as, e.g., TRIM24AFAP1, or PAN3). The fragment may comprise 8, 10,12, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 75, or more amino acids spoiling the fusion junction.

In certain embodiments, a fusion includes an in-frame fusion of all or aportion of the gene TRIM24 (e.g., a TRIM24 promoter or a functionalfragment thereof and one or more exons encoding TRIM24 or a fragmentthereof) and an exon of the NTRK2 gene (e.g., one or more exons encodingan NTRK2 kinase domain or a functional fragment thereof). Such a fusioncan be referred to as a TRIM24:NTRK2 fusion. In one embodiment, theTRIM24:NTRK2 fusion comprises sufficient TRIM24 and sufficient NTRK2sequences to drive expression of a fusion protein that has kinaseactivity. In some embodiments, the TRIM24:NTRK2 fusion drives expressionof a fusion protein that has elevated activity as compared with wildtype NTRK2 in the same tissue or cell.

In a particular embodiment, the invention provides a TRIM24:NTRK2 genefusion comprising the nucleotide sequence depicted in FIG. 1 (SEQ IDNO:1), or a fragment thereof that includes the fusion junction. SEQ IDNO:1 comprises TRIM24 (NM_015905) up to exon number 12 (amino acidnumber 671) fused to NTRK2 (NM_006180), from exon number 16 (amino acidnumber 483). In some embodiments the TRIM24:NTRK2 gene fusion comprisesa nucleotide sequence that is at least 85%, at least 90%, at least 95%,at least 97%, at least 98%, or at least 99% identical to all or part ofSEQ ID NO: 1. In another particular embodiment, the TRIM24:NTRK2 fusionhas the nucleotide sequence depicted in FIG. 2 (SEQ ID NO:2), comprisingTRIM24 (NM_015905) up to exon number 12 (amino acid number 671) fused toNTRK2 (NM_006180), from exon number 15 (amino acid number 467) or afragment thereof that includes the fusion junction. In some embodiments,the TRIM24:NTRK2 gene fusion comprises a nucleotide sequence that is atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% identical to all or part of SEQ ID NO:2.

In one embodiment, the TRIM24:NTRK2 gene fusion encodes a protein havingall or part of the sequence depicted in FIG. 3 (SEQ ID NO:3) or asequence that is at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% identical to all or part of SEQ ID NO:3.In another embodiment, the TRIM24:NTRK2 fusion encodes a protein havingall or part the sequence depicted in FIG. 4 (SEQ ID NO:4) or a sequencethat is at least 85%, at least 90%, at least 95%, at least 97%, at least98%, or at least 99% identical to all or part of SEQ ID NO:4.

In some embodiments, a fusion includes an in-frame fusion of all or aportion of gene AFAP1 (e.g., an AFAP1 promoter or a functional fragmentthereof and one or more exons encoding AFAP1 or a fragment thereof) andan exon of gene NTRK2 (e.g., one or more exons encoding an NTRK2 kinasedomain or a functional fragment thereof). Such a fusion can be referredto as an AFAP1:NTRK2 fusion. In one embodiment, the AFAP1:NTRK2 fusioncomprises sufficient AFAP1 and sufficient NTRK2 sequences to driveexpression of a fusion protein that has kinase activity. In someembodiments, the AFAP1:NTRK2 fusion drives expression of a fusionprotein that has elevated activity as compared with wild type NTRK2 inthe same tissue or cell.

In a particular embodiment, the AFAP1:NTRK2 fusion has the nucleotidesequence depicted in FIG. 5 (SEQ ID NO:5), comprising AFAP1 (NM_198595)up to exon number 13 (amino acid number 548) fused to NTRK2 (NM_006180),from exon number 12 (amino acid number 388) or a fragment thereof thatincludes the fusion junction. In some embodiments the AFAP1:NTRK2 genefusion comprises a nucleotide sequence that is at least 85%, at least90%, at least 95%, at least 97%, at least 98%, or at least 99% identicalto all or part of SEQ ID NO:5. In one embodiment, the AFAP1:NTRK2 fusionencodes a protein having all or part of the sequence depicted in FIG. 6(SEQ ID NO:6) or a sequence that is at least 85%, at least 90%, at least95%, at least 97%, at least 98%, or at least 99% identical to all orpart of SEQ ID NO:6.

In yet other embodiments, a fusion includes an in-frame fusion of all ora portion of gene PAN3 (e.g., a PAN3 promoter or a functional fragmentthereof and one or more exons encoding PAN3 or a fragment thereof) andan exon of gene NTRK2 (e.g., one or more exons encoding an NTRK2 kinasedomain or a functional fragment thereof). Such a fusion can be referredto as a PAN3:NTRK2 fusion. In one embodiment, the PAN3:NTRK2 fusioncomprises sufficient PAN3 and sufficient NTRK2 sequences to driveexpression of a fusion protein that has kinase activity. In someembodiments, the PAN3:NTRK2 fusion drives expression of a fusion proteinthat has elevated activity as compared with wild type NTRK2 in the sametissue or cell.

In a particular embodiment, the PAN3:NTRK2 fusion has the nucleotidesequence depicted in FIG. 7 (SEQ ID NO:7), comprising PAN3 (NM_175854)up to exon number 1 (amino acid number 143) fused to NTRK2 (NM_006180),from exon number 17 (amino acid number 546) or a fragment thereof thatincludes the fusion junction. In some embodiments the PAN3:NTRK2 genefusion comprises a nucleotide sequence that is at least 85%, at least90%, at least 95%, at least 97%, at least 98%, or at least 99% identicalto all or part of SEQ ID NO:7. In one embodiment, the PAN3:NTRK2 fusionencodes a protein having all or part of the sequence depicted in FIG. 8(SEQ ID NO:8) or a sequence that is at least 85%, at least 90%, at least95%, at least 97%, at least 98%, or at least 99% identical to all orpart of SEQ ID NO:8.

The nucleic acid sequences of NTRK2 gene fusions may be used as probes,primers, or bait to identify nucleotides from a biological sample thatinclude, flank, or hybridize to NTRK2 fusions, such as TRIM24:NTRK2 (forexample, all or part of SEQ ID NO:1 or SEQ ID NO:2), AFAP1:NTRK2 (forexample, all or part of SEQ ID NO:5), or PAN3:NTRK2 (for example, all orpart of SEQ ID NO:7), at, e.g., the fusion junctions. In certainembodiments, the probe, primer, or bait molecule is an oligonucleotidethat allows capture, detection, and/or isolation of an NTRK2 gene fusionin a biological sample. In certain embodiments, the probes or primersderived from the nucleic acid sequences of NTRK2 gene fusions (e.g.,from the fusion junctions) may be used, for example, for polymerasechain reaction (PCR) amplification. The oligonucleotide can comprise anucleotide sequence substantially complementary to a fragment of theNTRK2 gene fusion nucleic acid molecules described herein. The sequenceidentity between the nucleic acid fragment, e.g., the oligonucleotideand the target NTRK2 gene fusion sequence, need not be exact, so long asthe sequences are sufficiently complementary to allow the capture,detection, and/or isolation of the target sequence. In one embodiment,the nucleic acid fragment is a probe or primer that includes anoligonucleotide between about 5 and 25, e.g., between 10 and 20, or 10and 15 nucleotides in length that includes the fusion junction of anNTRK2 fusion, such as, e.g., TRIM24:NTRK2 (for example, all or part ofSEQ ID NO:1 or SEQ ID NO:2), AFAP1:NTRK2 (for example, all or part ofSEQ ID NO:5), or PAN3:NTRK2 (for example, all or part of SEQ ID NO:7).In other embodiments, the nucleic acid fragment is a bait that includesan oligonucleotide between about 100 to 300 nucleotides, 130 and 230nucleotides, or 150 and 200 nucleotides in length that includes thefusion junction of an NTRK2 fusion, such as, e.g., TRIM24:NTRK2 (forexample, all or part of SEQ ID NO:1 or SEQ ID NO:2), AFAP1:NTRK2 (forexample, all or part of SEQ ID NO:5), or PAN3:NTRK2 (for example, all orpart of SEQ ID NO:7).

In certain embodiments, the nucleic acid fragments hybridize to anucleotide sequence that includes a breakpoint or fusion junction, e.g.,a breakpoint or fusion junction as identified by a slash (“/”) in FIGS.1, 2, 5, and 7. For example, the nucleic acid fragment can hybridize toa nucleotide sequence that includes the fusion junction between theTRIM24 transcript and the NTRK2 transcript (e.g., nucleotides 2014-2016of SEQ NO:1, or nucleotides 2004-2006 of SEQ ID NO:2), or between theAFAP1 transcript and the NTRK2 transcript (e.g., nucleotides 1645-4647of SEQ ID NO:5), or between the PAN3 transcript and the NTRK2 transcript(e.g., nucleotides 430-432 of SEQ ID NO:7), i.e., a nucleotide sequencethat includes a portion of SEQ ID NO: 1, 2, 5, or 7. Examples include anucleotide sequence within exons 1 to 12 of a TRIM24 gene and exons 15or 16 to 22 of an NTRK2 gene (e.g., a portion of SEQ NO:1 comprisingnucleotides 2010-2019, 2005-2024, 1990-2039, 1965-2064, 1940-2089, or1915-2114; or a portion of SEQ ID NO:2 comprising nucleotides 2000-2009,1995-2014, 1980-2029, 1955-2054, 1930-2079, or 1905-2104); a nucleotidesequence within exons 1 to 13 of an AFAP1 gene and exons 12 to 22 of anNTRK2 gene (e.g., a portion of SEQ ID NO:5 comprising nucleotides1641-1650, 1636-1655, 1621-1670, 1596-1695, 1571-1720, or 1546-1745);and a nucleotide sequence within exon 1 of a PAN3 gene and exons 17-22of an NTRK2 gene (e.g., the portion of SEQ ID NO:7 comprisingnucleotides 426-435, 421-440, 406-455, 381-480, 354-504, or 330-529).

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to an NTRK2 gene fusionnucleic acid molecule described herein, and thereby allows thedetection, capture, and/or isolation of the nucleic acid molecule. Inone embodiment, a bait is suitable for solution phase hybridization. Inother embodiments, a bait includes a binding entity or detection entity,e.g., an affinity tag or fluorescent label, that allows detection,capture, and/or separation, e.g., by binding to a binding entity, of ahybrid formed by a bait and a nucleic acid hybridized to the bait.

In exemplary embodiments, the nucleic acid fragments used as baitcomprise a nucleotide sequence that includes a fusion junction betweenthe TRIM24 transcript and the NTRK2 transcript, e.g., a nucleotidesequence within SEQ ID NO:1 comprising nucleotides 2014-2016 (such as,e.g., a sequence comprising nucleotides 2010-2019, 2005-2024, 1990-2039,1965-2064, 1940-2089, or 1915-2114 of SEQ ID NO:1) or a nucleotidesequence within SEQ ID NO:2 comprising nucleotides 2004-2006 (such as,e.g., a sequence comprising nucleotides nucleotides 2000-2009,1995-2014, 1980-2029, 1955-2054, 1930-2079, or 1905-2104 of SEQ IDNO:2). In another exemplary embodiment, the nucleic acid sequenceshybridize to a nucleotide sequence that includes a fusion junctionbetween the AFAP1 transcript and the NTRK2 transcript, e.g., anucleotide sequence within SEQ ID NO:5 comprising nucleotides 1645-1647(such as, e.g., a sequence comprising nucleotides 1641-1650, 1636-1655,1621-1670, 1596-1695, 1571-1720, or 1546-1745 of SEQ ID NO:5). Inanother exemplary embodiment, the nucleic acid sequences hybridize to anucleotide sequence that includes a fusion junction between the PAN3transcript and the NTRK2 transcript, e.g., a nucleotide sequence withinSEQ ID NO:7 comprising nucleotides 430-432 (such as, e.g., a sequencecomprising nucleotides 426-435, 421-440, 406-455, 381-480, 354-504, or330-529 of SEQ ID NO:7).

Another aspect of the invention provides NTRK2 fusion proteins (such as,e.g., a purified or isolated TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2fusion protein), biologically active or antigenic fragments thereof, anduse of those polypeptides for detecting and/or modulating the biologicalactivity (such as tumorigenic activity) of an NTRK2 fusion protein.Exemplary embodiments of the NTRK2 fusion proteins comprise the aminoacid sequence set forth in SEQ ID NO:3, 4, 6, or 8, and fragments ofthose sequences.

In some embodiments, the NTRK2 fusion protein of the invention includesa fragment of a TRIM24 protein, an AFAP1 protein, or a PAN3 protein anda fragment of an NTRK2 protein. In one embodiment, the NTRK2 fusionprotein is TRIM24:NTRK2 fusion protein having the amino acid sequence ofSEQ ID NO:3 or a fragment thereof, such as, e.g., amino acids 670-674,665-674, 662-681, or 652-691 of SEQ ID NO:3. In another embodiment, theNTRK2 fusion protein is a TRIM24:NTRK2 fusion protein having the aminoacid sequence of SEQ ID NO:4 or a fragment thereof, such as, e.g., aminoacids 670-674, 665-674, 662-681, or 652-691 of SEQ ID NO:4. In oneembodiment, the NTRK2 fusion protein is an AFAP1:NTRK2 fusion proteinhaving the amino acid sequence of SEQ ID NO:6 or a fragment thereof,such as, e.g., amino acids 547-551, 544-553, 539-558, or 529-568 of SEQID NO:6. In another embodiment, the NTRK2 fusion protein is a PAN3:NTRK2fusion protein having the amino acid sequence of SEQ ID NO:8 or afragment thereof, such as, e.g., amino acids 142-146, 138-147, 133-152,or 123-162 of SEQ ID NO:8.

In yet another embodiment, the NTRK2 fusion protein is a TRIM24:NTRK2fusion protein comprising an amino acid sequence that is at least 35%,at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO:3 or a fragment thereof (e.g., amino acids670-674, 665-674, 662-681, or 652-691 of SEQ ID NO:3). In anotherembodiment, the NTRK2 fusion protein is a TRIM24:NTRK2 fusion proteincomprising an amino acid sequence that is at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:4 or a fragment thereof (e.g., amino acids 670-674, 665-674,662-681, or 652-691 of SEQ ID NO:4). In yet another embodiment, theNTRK2 fusion protein is an AFAP1:NTRK2 fusion protein comprising anamino acid sequence that is at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% identical to all or part of SEQID NO:6 (e.g., amino acids 547-551, 544-553, 539-558, or 529-568 of SEQID NO:6). In another embodiment the NTRK2 fusion protein is a PAN1:NTRK2fusion protein comprising an amino acid sequence that is at least 85%,at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to SEQ NO:8 or a fragment thereof (e.g., amino acids 142-146,138-147, 133-152, or 123-162 of SEQ ID NO:8).

In certain embodiments, the NTRK2 fusion protein includes a functionalkinase domain. In such embodiments, the NTRK2 fusion protein compriseselevated NTRK2 activity as compared with wild type NTRK2, for example,in a cancer cell, a non-cancer cell adjacent to the cancer cell, or anon-cancer cell from a control sample, such as a cancer free subject.Ira one exemplary embodiment, the NTRK2 fusion protein is a TRIM24:NTRK2fusion and includes an NTRK2 tyrosine kinase domain or a functionalfragment thereof. In another exemplary embodiment, the NTRK2 fusionprotein is an AFAP1:NTRK2 fusion and includes an NTRK2 tyrosine kinasedomain or a functional fragment thereof. In yet another embodiment, theNTRK2 fusion protein is a PAN3:NTRK2 fusion and includes an NTRK2tyrosine kinase domain or a functional fragment thereof.

In another embodiment, the NTRK2 fusion protein or fragment is apeptide, e.g., an immunogenic peptide or protein, that contains a fusionjunction with a heterologous protein as described herein. Suchimmunogenic peptides or proteins can be used for vaccine preparation foruse in the treatment or prevention of cancers cause by or exacerbated byNTRK2 gene fusions and NTRK2 fusion proteins. In other embodiments, suchimmunogenic peptides or proteins can be used to raise antibodiesspecific to the fusion protein. In some embodiments, the NTRK2 fusionprotein is present in combination with or is further conjugated to oneor more adjuvant(s) or immunogen(s), e.g., a protein capable ofenhancing an immune response to the NTRK2 fusion protein (e.g., ahapten, a toxoid, etc.). In some embodiments, the NTRK2 fusion proteinis a TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 fusion. In someembodiments, the NTRK2 fusion protein comprises the fusion junction ofSEQ ID NO:3, 4, 6, or 8.

Thus, another aspect of the invention provides an antibody that binds toan NTRK2 fusion protein (such as, e.g., a TRIM24:NTRK2, an AFAP1:NTRK2,or a PAN3:NTRK2 fusion protein) or a fragment thereof. In certainembodiments, the antibody recognizes an NTRK2 fusion protein but doesnot recognize wild type NTRK2 or the wild type fusion partner (such as,e.g., TRIM24, AFAP1, or PAN3). In some embodiments, the antibody bindsto an epitope comprising the fusion junction between NTRK2 and thefusion partner (e.g., the fusion junction of TRIM24:NTRK2, AFAP1:NTRK2,or PAN3:NTRK2). In one embodiment, the antibody binds to a TRIM24:NTRK2fusion protein having the amino acid sequence of SEQ ID NO:3 or afragment thereof, such as, e.g., amino acids 670-674, 665-674, 662-681,or 652-691 of SEQ ID NO:3. In one embodiment, the antibody binds to aTRIM24:NTRK2 fusion protein having the amino acid sequence of SEQ IDNO:4 or a fragment thereof, such as, e.g., amino acids 670-674, 665-674,662-681, or 652-691 of SEQ ID NO:4. In other embodiments, the antibodybinds to an AFAP1:NTRK2 fusion protein having the amino acid sequence ofSEQ ID NO:6 or a fragment thereof, such as, e.g., amino acids 547-551,544-553, 539-558, or 529-568 of SEQ ID NO:6. In yet other embodiments,the antibody binds to a PAN3:NTRK2 fusion protein having the amino acidsequence of SEQ ID NO:8 or a fragment thereof, such as, e.g., aminoacids 142-146, 138-147, 133-152, or 123-162 of SEQ ID NO:8.

In certain embodiments, the antibodies of the invention inhibit and/orneutralize the biological activity of the NTRK2 fusion protein, and morespecifically, in some embodiments, the kinase activity of the NTRK2fusion protein. In other embodiments, the antibodies may be used todetect an NTRK2 fusion protein or to diagnose a patient suffering from adisease or disorder associated with the expression of an NTRK2 fusionprotein.

Detection and Diagnostic Methods

In another aspect, the invention provides a method of determining thepresence of an NTRK2 gene fusion or fusion protein, such as, e.g., aTRIM124:NTRK2, an AFAP1:NTRK2, or a PAN3:NTRK2 fusion as describedherein. The presence of an NTRK2 gene fusion can indicate that themammal providing the biological sample suffers from or is at risk ofdeveloping a disorder mediated by aberrant NTRK2 expression or activity,or overexpression of NTRK2, such as, e.g., a cancer. The presence of anNTRK2 gene fusion may also indicate that the cancer is treatable with anNTRK2 inhibitor (such as, e.g., an antibody specific to NTRK2) or anNTRK2 fusion inhibitor. In some embodiments the cancer is lung cancer.In some embodiments, the cancer is lung adenocarcinoma. In someembodiments the cancer is a glioma. In some embodiments, the cancer is alow grade glioma. In some embodiments, the cancer is squamous carcinoma.In some embodiments, the cancer is head and neck squamous cellcarcinoma. In other embodiments, the cancer is a different cancerassociated with aberrant expression or activity of NTRK2 oroverexpression of NTRK2.

In one embodiment, the NTRK2 fusion detected is a nucleic acid moleculeor a polypeptide. The method includes detecting whether an NTRK2 fusionnucleic acid molecule or polypeptide is present in a cell (e.g., acirculating cell or a cancer cell), a tissue (e.g., a tumor), or asample (e.g., a tumor sample), from a subject. In one embodiment, thesample is a nucleic acid sample. In one embodiment, the nucleic acidsample comprises DNA, e.g., genomic DNA or cDNA, or RNA, e.g., mRNA. Inother embodiments, the sample is a protein sample. The sample can bechosen from one or more of sample types: such as, e.g., tissue, e.g.,cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma,buccal scrape, sputum, saliva, cerebrospinal fluid, urine, stool,circulating tumor cells, circulating nucleic acids, or bone marrow.

In some embodiments, the NTRK2 fusion is detected in a nucleic acidmolecule by one or more methods chosen from nucleic acid hybridizationassays (e.g. in situ hybridization, comparative genomic hybridization,microarray, Southern blot, northern blot), amplification-based assays(e.g., PCR, PCR-RFLP assay, or real-time PCR), sequencing and genotyping(e.g. sequence-specific primers, high-performance liquid chromatography,or mass-spectrometric genotyping), and screening analysis (includingmetaphase cytogenetic analysis by karyotype methods).

Hybridization Methods

In some embodiments, the reagent hybridizes to an NTRK2 gene fusion,such as, e.g., nucleotides 2014-2016, 2010-2019, 2005-2024, 1990-2039,1965-2064, 1940-2089, or 1915-2114 of SEQ ID NO: 1. In alternateembodiments, the reagent detects the presence of nucelotides 2004-2006,2000-2009, 1995-2014, 1980-2029, 1955-2054, 1930-2079, or 1905-2104 ofSEQ ID NO:2, nucleotides 1645-1647, 1641-1650, 1636-1655, 1621-1670,1596-1695, 1571-1720, or 1546-1745 of SEQ ID NO:5, or nucleotides430-432, 426-435, 421-440, 406-455, 381-480, 354-504, or 330-529 of SEQID NO:7. In an alternate embodiment, the method includes the steps ofobtaining a sample; exposing the sample to a nucleic acid probe whichhybridizes to an mRNA or cDNA encoding an NTRK2 fusion protein thatcomprises amino acids 670-674, 665-674, 662-681, or 652-691 of SEQ IDNO:3, amino acids 670-674, 665-674, 662-681, or 652-691 of SEQ ID NO:4,amino acids 547-551, 544-553, 539-558, or 529-568 of SEQ ID NO:6, oramino acids 142-146, 138-147, 133-152, or 123-162 of SEQ ID NO:8,wherein hybridization of the probe to the mRNA or cDNA in the sampleindicates the presence of an NTRK2 fusion polynucleotide.

Hybridization, as described throughout the specification, may be carriedout under stringent conditions, e.g., medium or high stringency. See,e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Pr; 2nd edition (1989);T. Brown. Hybridization Analysis of DNA Blots. Current Protocols inMolecular Biology at 21:2.10.1 -2.10.16 (2001). High stringencyconditions for hybridization refer to conditions under which two nucleicacids must possess a high degree of base pair homology to each other inorder to hybridize. Examples of highly stringent conditions forhybridization include hybridization in 4*sodium chloride/sodium citrate(SSC), at 65 or 70° C., or hybridization in 4*SSC plus 50% formamide atabout 42 or 50° C., followed by at least one, at least two, or at leastthree washes in 1*SSC, at 65 or 70° C. Another example of highlystringent conditions includes hybridization in 2*SSC; 10*Denhardtsolution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mMNa₂HPO₄; 250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or 0.25 M ofsodium phosphate buffer, pH 7.2; 1 mM EDTA 7% SDS at 60° C; followed bywashing 2*SSC, 0.1% SDS at 60° C.

The nucleic acid fragments can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label (e.g.,biotin/streptavidin), or can include an affinity tag or identifier(e.g., an adaptor, barcode or other sequence identifier). Labeled orunlabeled nucleic acids and/or nucleic acid fragments may be used inreagents for detecting, capturing, and/or isolating NTRK2 gene fusions,such as, e.g., TRIM24:NTRK2 (for example, all or part of SEQ ID NO:1 orSEQ ID NO:2), AFAP1:NTRK2 (for example, all or part of SEQ ID NO:5), orPAN3:NTRK2 (for example, all or part of SEQ ID NO:1 or SEQ ID NO:7).

In some embodiments, the method comprises performing chromosome in situhybridization with chromosomal DNA from a biological sample to detectthe presence of an NTRK2 gene fusion (such as, e.g., TRIM24:NTRK2,AFAP1:NTRK2, or PAN3:NTRK2, as disclosed herein). In some embodiments,the chromosome in situ hybridization comprises the steps of: providing achromosome (e.g., interphase or metaphase chromosome) preparation (e.g.,by attaching the chromosomes to a substrate (e.g., glass)); denaturingthe chromosomal DNA (e.g., by exposure to formamide) to separate thedouble strands of the polynucleotides from each other; exposing thenucleic acid probe to the chromosomes under conditions to allowhybridization of the probe to the target DNA; removing unhybridized ornon-specifically hybridized probes by washing; and detecting thehybridization of the probe with the target DNA. In some embodiments, thechromosome in situ hybridization is fluorescence in situ hybridization(FISH). In some embodiments, the probe is labeled directly by afluorescent label, or indirectly by incorporation of a nucleotidecontaining a tag or reporter molecule (e.g., biotin, digoxigenin, orhapten) which after hybridization to the target DNA is then bound byfluorescently labeled affinity molecule (e.g., an antibody orstreptavidin). In some embodiments, the hybridization of the probe withthe target DNA in FISH can be visualized using a fluorescencemicroscope.

In other embodiments, the method comprises performing Southern blot withDNA polynucleotides from a biological sample to detect the presence ofan NTRK2 gene fusion (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, orPAN3:NTRK2, as disclosed herein). In some embodiments, the Southern blotcomprises the steps of: optionally fragmenting the polynucleotides intosmaller sizes by restriction endonucleases; separating thepolynucleotides by gel electrophoresis; denaturing the polynucleotides(e.g., by heat or alkali treatment) to separate the double strands ofthe polynucleotides from each other; transferring the polynucleotidesfrom the gel to a membrane (e.g., a nylon or nitrocellulose membrane);immobilizing the polynucleotides to the membrane (e.g., by UV light orheat); exposing the nucleic acid probe to the polynucleotides underconditions to allow hybridization of the probe to the target DNA;removing unhybridized or non-specifically hybridized probes by washing;and detecting the hybridization of the probe with the target DNA.

Amplification-Based Assays

In certain embodiments, the method of detecting the presence of an NTRK2gene fusion, comprises (a) performing a PCR amplification reaction withpolynucleotides from a biological sample, wherein the amplificationreaction utilizes a pair of primers which will amplify at least afragment of the NTRK2 gene fusion, wherein the fragment comprises thefusion junction, wherein the first primer is in sense orientation andthe second primer is in antisense orientation; and (b) detecting anamplification product, wherein the presence of the amplification productis indicative of the presence of an NTRK2 fusion polynucleotide in thesample. In specific exemplary embodiments, the NTRK2 gene fusion isTRIM24:NTRK2, such as, e.g., the gene fusion of SEQ ID NO: 1, or SEQ IDNO:2, or a fragment thereof, e.g., a nucleotide sequence comprisingnucleotides 2014-2016, 2010-2019, 2005-2024, 1990-2039, 1965-2064,1940-2089, or 1915-2114 of SEQ ID NO:1 or nucleotides 2004-2006,2000-2009, 1995-2014, 1980-2029, 1955-2054, 1930-2079, or 1905-2104 ofSEQ ID NO:2. In other exemplary embodiments, the gene fusion isAFAP1:NTRK2 such as, e.g. the gene fusion of SEQ ID NO:5 or a fragmentthereof, e.g., a nucleotide sequence comprising nucleotides 1645-1647,1641-1650, 1636-1655, 1621-1670, 1596-1695, 1571-1720, or 1546-1745 ofSEQ ID NO:5. In some exemplary embodiments, the gene fusion isPAN3:NTRK2 such as, e.g. the gene fusion of SEQ ID NO:7 or a fragmentthereof, e.g., a nucleotide sequence comprising nucleotides 430-432,426-435, 421-440, 406-455, 381-480, 354-504, or 330-529 of SEQ ID NO:7.In some embodiments, step (a) of performing a PCR amplification reactioncomprises: (i) providing a reaction mixture comprising thepolynucleotides (e.g., DNA or cDNA) from the biological sample, the pairof primers which will amplify at least a fragment of the NTRK2 genefusion wherein the first primer is complementary to a sequence on thefirst strand of the polynucleotides and the second primer iscomplementary to a sequence on the second strand of the polynucleotides,a DNA polymerase, and a plurality of free nucleotides comprisingadenine, thymine, cytosine, and guanine (dNTPs); (ii) heating thereaction mixture to a first predetermined temperature for a firstpredetermined time to separate the double strands of the polynucleotidesfrom each other; (iii) cooling the reaction mixture to a secondpredetermined temperature for a second predetermined time underconditions to allow the first and second primers to hybridize with theircomplementary sequences on the first and second strands of thepolynucleotides, and to allow the DNA polymerase to extend the primers;and (iv) repeating steps (ii) and (iii) for a predetermined number ofcycles (e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles). In someembodiments, the polynucleotides from the biological sample compriseRNA, and the method further comprises performing a RT-PCR amplificationreaction with the RNA to synthesize cDNA as the template for subsequentor simultaneous PCR reactions. In some embodiments, the RT-PCRamplification reaction comprises providing a reaction mixture comprisingthe RNA, a primer which will amplify a fragment of the RNA (e.g., asequence-specific primer, a random primer, or oligo(dT)s), a reversetranscriptase, and dNTPs, and heating the reaction mixture to a thirdpredetermined temperature for a third predetermined time underconditions to allow the reverse transcriptase to extend the primer.

Sequencing and Genotyping

Another method for determining the presence of an NTRK2 gene fusionmolecule (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, asdisclosed herein) includes: sequencing a portion of the nucleic acidmolecule (e.g., sequencing the portion of the nucleic acid molecule thatcomprises the fusion junction of an NTRK2 gene fusion), therebydetermining that the NTRK2 gene fusion is present in the nucleic acidmolecule. In some exemplary embodiments, the gene fusion isTRIM24:NTRK2. In other exemplary embodiments, the gene fusion isAFAP1:NTRK2. In yet other exemplary embodiments, the gene fusion isPAN3:NTRK2. Optionally, the sequence acquired is compared to a referencesequence, or a wild type reference sequence. In one embodiment, thesequence is determined by a next generation sequencing method. In someembodiments, the sequencing is automated and/or high-throughputsequencing. The method can further include acquiring, e.g., directly orindirectly acquiring, a sample, e.g., a tumor or cancer sample, from apatient.

In some embodiments, the sequencing comprises chain terminatorsequencing (Sanger sequencing), comprising: providing a reaction mixturecomprising a nucleic acid molecule from a biological sample, a primercomplementary to a region of the template nucleic acid molecule, a DNApolymerase, a plurality of free nucleotides comprising adenine, thymine,cytosine, and guanine (dNTPs), and at least one chain terminatingnucleotide (e.g., at least one di-deoxynucleotide (ddNTPs) chosen fromddATP, ddTTP, ddTTP, and ddGTP), wherein the at least one chainterminating nucleotide is present in a low concentration so that chaintermination occurs randomly at any one of the positions containing thecorresponding base on the DNA strand; annealing the primer to a singlestrand of the nucleic acid molecule; extending the primer to allowincorporation of the chain terminating nucleotide by the DNA polymeraseto produce a series of DNA fragments that are terminated at positionswhere that particular nucleotide is used; separating the polynucleotidesby electrophoresis (e.g., gel or capillary electrophoresis); anddetermining the nucleotide order of the template nucleic acid moleculebased on the positions of chain termination on the DNA fragments. Insome embodiments, the sequencing is carried out with four separatebase-specific reactions, wherein the primer or the chain terminatingnucleotide in each reaction is labeled with a separate fluorescentlabel. In other embodiments, the sequencing is carried out in a singlereaction, wherein the four chain terminating nucleotides mixed in thesingle reaction are each labeled with a separate fluorescent label.

In some embodiments, the sequencing comprises pyrosequencing (sequencingby synthesis), comprising: (i) providing a reaction mixture comprising anucleic acid molecule from a biological sample, a primer complementaryto a region of the template nucleic acid molecule, a DNA polymerase, afirst enzyme capable of converting pyrophosphate into ATP, and a secondenzyme capable using ATP to generates a detectable signal (e.g., achemiluminescent signal, such as light) in an amount that isproportional to the amount of ATP; (ii) annealing the primer to a singlestrand of the nucleic acid molecule; (iii) adding one of the four freenucleotides (dNTPs) to allow incorporation of the correct, complementarydNTP) onto the template by the DNA polymerase and release ofpyrophosphate stoichiometrically; (iv) converting the releasedpyrophosphate to ATP by the first enzyme; (v) generating a detectablesignal by the second enzyme using the ATP; (vi) detecting the generatedsignal and analyzing the amount of signal generated in a pyrogram; (vii)removing the unincorporated nucleotides; and (viii) repeating steps(iii) to (vii). The method allows sequencing of a single strand of DNA,one base pair at a time, and detecting which base was actually added ateach step. The solutions of each type of nucleotides are sequentiallyadded and removed from the reaction. Light is produced only when thenucleotide solution complements the first unpaired base of the template.The order of solutions which produce detectable signals allows thedetermination of the sequence of the template.

In some embodiments, the method of determining the presence of an NTRK2fusion (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, asdisclosed herein) comprises analyzing a nucleic acid sample (e.g., DNA,cDNA, or RNA, or an amplification product thereof) by HPLC. The methodmay comprise: passing a pressurized liquid solution containing thesample through a column filled with a sorbent, wherein the nucleic acidor protein components in the sample interact differently with thesorbent, causing different flow rates for the different components;separating the components as they flow out the column at different flowrates. In some embodiments, the HPLC is chosen from, e.g., reverse-phaseHPLC, size exclusion HPLC, ion-exchange HPLC, and bioaffinity HPLC.

In some embodiments, the method of determining the presence of an NTRK2fusion (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, asdisclosed herein) comprises analyzing a nucleic acid sample (e.g., DNA,cDNA, or RNA, or an amplification product thereof) by mass spectrometry.The method may comprise: ionizing the components in the sample (e.g., bychemical or electron ionization); accelerating and subjecting theionized components to an electric or magnetic field; separating theionized components based on their mass-to-charge ratios; and detectingthe separated components by a detector capable of detecting chargedparticles (e.g., by an electron multiplier).

Methods for Detecting Fusion Proteins

Another aspect of the invention provides a method of determining thepresence of an NTRK2 fusion protein (such as, e.g., TRIM24:NTRK2,AFAP1:NTRK2, or PAN3:NTRK2, as disclosed herein) in a mammal. The methodcomprises the steps of obtaining a biological sample of a mammal (suchas, e.g., from a human cancer ), and exposing that sample to at leastone reagent that detects an NTRK2 fusion protein (e.g., an antibody thatrecognizes the NTRK2 fusion but does not recognize the wild type NTRK2or the wild type fusion partner) to determine whether an NTRK2 fusionprotein is present in the biological sample. The detection of an NTRK2fusion protein indicates the presence of a mutant NTRK2 in the mammal(such as, e.g., in the human cancer). In some embodiments, the NTRK2fusion protein comprises an amino acid sequence having at least 85%,90%, 95%, 97%, 98%, or 99% identity with an amino acid sequence of anyone of SEQ ID NOs 3, 4, 6, and 8. In some embodiments the cancer is lungcancer, such as, e.g. lung adenocarcinoma. In some embodiments, thecancer is a glioma, such as, e.g. a low grade glioma. In someembodiments, the cancer is squamous cell carcinoma, such as, e.g., headand neck squamous cell carcinoma. In some embodiments, the reagent thatdetects an NTRK2 fusion protein can be delectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label (e.g.,biotin/streptavidin), an antigen label, or can include an affinity tagor identifier (e.g., an adaptor, barcode or other sequence identifier).In some embodiments, the labeled reagent can be detected using, e.g.,autoradiography, microscopy (e.g., brightfield, fluorescence, orelectron microscopy), ELISA, or immunohistochemistry. In someembodiments, the NTRK2 fusion protein is detected in a biological sampleby a method chosen from one or more of: antibody-based detection (e.g.,western blot, ELISA, immunohistochemistry), size-based detection methods(e.g., HPLC or mass spectrometry), or protein sequencing.

Antibody-Based Detection

In some embodiments, the method comprises performing a western blot withpolypeptides from a biological sample to detect the presence of an NTRK2fusion protein (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2,as disclosed herein). In some embodiments, the western blot comprisesthe steps of: separating the polypeptides by gel electrophoresis;transferring the polypeptides from the gel to a membrane (e.g., anitrocellulose or polyvinylidene difluoride (PVDF) membrane); blockingthe membrane to prevent nonspecific binding by incubating the membranein a dilute solution of protein (e.g., 3-5% bovine serum albumin (BSA)or non-fat dry milk in Tris-Buffered Saline (TBS) or I-Block, with aminute percentage (e.g., 0.1%) of detergent, such as, e.g., Tween 20 orTriton X-100); exposing the polypeptides to at least one reagent thatdetects an NTRK2 fusion protein (e.g., an antibody that recognizes theNTRK2 fusion but does not recognize the wild type NTRK2 or the wild typefusion partner); removing unbound or non-specifically bound reagent bywashing; and detecting the binding of the reagent with the targetprotein. In some embodiments, the method comprises two-step detection:exposing the polypeptides to a primary antibody that specifically bindsto an NTRK2 fusion protein; removing unbound or non-specifically boundprimary antibody by washing; exposing the polypeptides to a secondaryantibody that recognizes the primary antibody; removing unbound ornon-specifically bound secondary antibody by washing; and detecting thebinding of the secondary antibody. In some embodiments, the reagent thatdetects an NTRK2 fusion protein (e.g., the fusion specific antibody, orthe secondary antibody) is directly labeled for detection. In otherembodiments, the reagent is linked to an enzyme, and the method furthercomprises adding a substrate of the enzyme to the membrane; anddeveloping the membrane by detecting a detectable signal produced by thereaction between the enzyme and the substrate. For example, the reagentmay be linked with horseradish peroxidase to cleave a chemiluminescentagent as a substrate, producing luminescence in proportion to the amountof the target protein for detection.

In some embodiments, the method comprises performing ELISA withpolypeptides from a biological sample to detect the presence of an NTRK2fusion protein (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2,as disclosed herein). In some embodiments, the ELISA is chosen from,e.g., direct ELISA, indirect ELISA, sandwich ELISA, and competitiveELISA.

In one embodiment, the direct ELISA comprises the steps of: attachingpolypeptides from a biological sample to a surface; blocking the surfaceto prevent nonspecific binding by incubating the surface in a dilutesolution of protein; exposing the polypeptides to an antibody thatspecifically binds to an NTRK2 fusion protein (e.g., an antibody thatrecognizes the NTRK2 fusion (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2,or PAN3:NTRK2, as disclosed herein) but does not recognize the wild typeNTRK2 or the wild type fusion partner); removing unbound ornon-specifically bound antibody by washing; and detecting the binding ofthe antibody with the target protein. In some embodiments, the antibodyis directly labeled for detection. In other embodiments, the antibody islinked to an enzyme, and the method further comprises adding a substrateof the enzyme; and detecting a detectable signal produced by thereaction between the enzyme and the substrate.

In another embodiment, the indirect ELISA comprises the steps of:attaching polypeptides from a biological sample to a surface; blockingthe surface to prevent nonspecific binding by incubating the surface ina dilute solution of protein; exposing the polypeptides to a primaryantibody that specifically binds to an NTRK2 fusion protein (such as,e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, as disclosed herein);removing unbound or non-specifically bound primary antibody by washing;exposing the polypeptides to a secondary antibody that recognizes theprimary antibody; removing unbound or non-specifically bound secondaryantibody by washing; and detecting the binding of the secondaryantibody. In some embodiments, the secondary antibody is directlylabeled for detection. In other embodiments, the secondary antibody islinked to an enzyme, and the method further comprises adding a substrateof the enzyme; and detecting a detectable signal produced by thereaction between the enzyme and the substrate.

In some embodiments, the method comprises performingimmunohistochemistry with polypeptides from a biological sample todetect the presence of an NTRK2 fusion protein (such as, e.g.,TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, as disclosed herein). In someembodiments, the immunohistochemistry comprises the steps of: fixing acell or a tissue section (e.g., by paraformaldehyde or formalintreatment); permeabilizing the cell or tissue section to allow targetaccessibility; blocking the cell or tissue section to preventnonspecific binding; exposing the cell or tissue section to at least onereagent that detects an NTRK2 fusion protein (e.g., an antibody thatrecognizes the NTRK2 fusion but does not recognize the wild type NTRK2or the wild type fusion partner); removing unbound or non-specificallybound reagent by washing; and detecting the binding of the reagent withthe target protein. In some embodiments, the reagent is directly labeledfor detection. In other embodiments, the reagent is linked to an enzyme,and the method further comprises adding a substrate of the enzyme; anddetecting a detectable signal produced by the reaction between theenzyme and the substrate. In some embodiments, the immunohistochemistrymay comprise the two-step detection as in the indirect ELISA.

Size-Based Detection Methods

In some embodiments, the method of determining the presence of an NTRK2fusion (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, asdisclosed herein) comprises analyzing a protein sample by HPLC. Themethod may comprise; passing a pressurized liquid solution containingthe sample through a column filled with a sorbent, wherein the nucleicacid or protein components in the sample interact differently with thesorbent, causing different flow rates for the different components;separating the components as they flow out the column at different flowrates. In some embodiments, the HPLC is chosen from, e.g., reverse-phaseHPLC, size exclusion HPLC, ion-exchange HPLC, and bioaffinity HPLC.

In some embodiments, the method of determining the presence of an NTRK2fusion (such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, asdisclosed herein) comprises analyzing a protein sample by massspectrometry. The method may comprise: ionizing the components in thesample (e.g., by chemical or electron ionization); accelerating andsubjecting the ionized components to an electric or magnetic field;separating the ionized components based on their mass-to-charge ratios;and detecting the separated components by a detector capable ofdetecting charged particles (e.g., by an electron multiplier).

Detection of an NTRK2 gene fusion or an NTRK2 fusion protein in apatient can lead to assignment of the patient to the newly identifiedpatient population that bears the NTRK2 fusion. Because this patientpopulation can suffer from or be susceptible to a disorder associatedwith aberrant NTRK2 expression or activity, or overexpression of NTRK2,detection of the NTRK2 fusion can also lead to diagnosis of suchdisorder. Thus, a further aspect of the invention provides a method ofstratifying a patient population (e.g., assigning a patient, to a groupor class) and/or diagnosing a patient, comprising: obtaining abiological sample from the patient, contacting the sample with at leastone reagent that detects an NTRK2 gene fusion or an NTRK2 fusion proteinto determine whether an NTRK2 fusion is present in the biologicalsample. The detection of an NTRK2 fusion indicates that the patientbelongs to the newly identified patient population that bears the NTRK2fusion, and/or the presence of a disorder associated with aberrant NTRK2expression or activity, or overexpression of NTRK2, such as, e.g.,certain cancers. The detection of a NTRK2 fusion also identifies a newsubtype of cancer, which is characterized by the presence of the NTRK2fusion, such as e.g., lung cancer (e.g., lung adenocarcinoma), glioma(e.g., low grade glioma), or squamous cell carcinoma (e.g., head andneck squamous cell carcinoma). In certain embodiments, the NTRK2 fusionis TRIM24:NTRK2. In some embodiments, the TRIM24:NTRK2 fusion has all orpart of the nucleotide and/or amino acid sequence (such as, e.g., thefusion junction) set forth in SEQ ID NO:1 and SEQ ID NO:3, respectively.In some embodiments, the TRIM24:NTRK2 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively. Inother embodiments, the NTRK2 fusion is AFAP1:NTRK2. In some embodiments,the AFAP1:NTRK2 fusion has all or part of the nucleotide and/or aminoacid sequence (such as, e.g., the fusion junction) set forth in SEQ IDNO:5 and SEQ ID NO:6, respectively, in yet other embodiments, the NTRK2fusion is PAN3:NTRK2. In some embodiments, the PAN3:NTRK2 fusion has allor part of the nucleotide and/or amino acid sequence (such as, e.g., thefusion junction) set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively.

In some embodiments, the NTRK2 gene fusion or NTRK2 fusion protein isdetected prior to initiating, during, and/or after, a treatment of apatient with, e.g., an NTRK2 inhibitor (such as, e.g., a kinaseinhibitor) or an NTRK2 fusion inhibitor. In one embodiment, the NTRK2gene fusion or NTRK2 fusion protein is detected at the time the patientis diagnosed with a cancer. In other embodiment, the. NTRK2 fusion isdetected at a pre-determined interval, e.g., a first point in time andat least at a subsequent point in time. In certain embodiments, inresponse to detection of an NTRK2 fusion, such as, e.g., TRIM24:NTRK2,AFAP1:NTRK2, or PAN3:NTRK2, the method further includes one or more of:

(1) stratifying a patient population (e.g., assigning a patient, to agroup or class);

(2) identifying or selecting the patient as likely or unlikely torespond to a treatment, e.g., a NTRK2 inhibitor treatment (e.g., akinase inhibitor treatment), or a NTRK2 fusion inhibitor treatment asdescribed herein;

(3) selecting a treatment regimen, e.g., administering or notadministering a preselected therapeutic agent, such as, e.g., an NTRK2inhibitor (e.g., a pan NTRK-1,2,3 inhibitor, or an NTRK2 specificinhibitor) or an NTRK2 fusion inhibitor;

(4) prognosticating the time course of the disease in the patient (e.g.,evaluating the likelihood of increased or decreased patient survival);or

(5) monitoring the effectiveness of treatment (e.g., by detecting areduction in the level of NTRK2 gene fusion or fusion protein in apatient sample).

In certain embodiments, upon detection of an NTRK2 gene fusion or NTRK2fusion protein in a patient's biological sample, the patient isidentified as likely to respond to a treatment that comprises an NTRK2inhibitor (e.g., a pan NTRK-1,2,3 inhibitor, or an NTRK2 specificinhibitor) or an NTRK2 fusion inhibitor. In some embodiments, the NTRK2fusion detected is a TRIM24:NTRK2 fusion. In alternate embodiments, theNTRK2 fusion detected is an AFAP1:NTRK2 fusion. In some embodiments, theNTRK2 fusion detected is a PAN3:NTRK2 fusion.

A further aspect of the invention provides a method of selecting atreatment option by detecting an NTRK2 fusion. The method comprisesobtaining a biological sample from a patient and exposing the sample toat least one reagent that detects an NTRK2 gene fusion or fusion proteinto determine whether an NTRK2 fusion is present in the biologicalsample. The detection of the NTRK2 gene fusion or fusion proteinindicates the likelihood of the patient responding to treatment with anNTRK inhibitor or an NTRK2 fusion inhibitor. The method may be augmentedor personalized by evaluating the effect of a variety of NTRK2inhibitors or NTRK2 fusion inhibitors on the biological sample shown tocontain an NTRK2 gene fusion or fusion protein to determine the mostappropriate inhibitor to administer. In certain embodiments, the NTRK2fusion is TRIM24:NTRK2. In some embodiments, the TRIM24:NTRK2 fusion hasall or part of the nucleotide and/or amino acid sequence (such as, e.g.,the fusion junction) set forth in SEQ ID NO:1 and SEQ ID NO:3,respectively. In some embodiments, the TRIM24:NTRK2 fusion has all orpart of the nucleotide and/or amino acid sequence (such as, e.g., thefusion junction) set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively.In other embodiments, the NTRK2 fusion is AFAP1:NTRK2. In someembodiments, the AFAP1:NTRK2 fusion has all or part of the nucleotideand/or amino acid sequence (such as, e.g., the fusion junction) setforth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In yet otherembodiments, the NTRK2 fusion is PAN3:NTRK2. In some embodiments, thePAN3:NTRK2 fusion has all or part of the nucleotide and/or amino acidsequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:7and SEQ ID NO:8, respectively.

Methods of Treatment

Alternatively, or in combination with the detection and diagnosticmethods described herein, the invention provides method for treating thenewly identified patient population and the new NTRK2 fusion cancersubtype, which are characterized by the presence of an NTRK2 fusion. Thepatient population and cancer subtype can be associated with or predictthe onset of a condition mediated by aberrant NTRK2 expression oractivity, or overexpression of NTRK2, such as, e.g., a cancer or a tumorharboring an NTRK2 fusion. In certain embodiments, the cancer or tumorharboring an NTRK2 fusion is lung cancer (e.g., lung adenocarcinoma),glioma (e.g., low grade glioma), or squamous cell carcinoma (e.g., headand neck squamous cell carcinoma). The methods comprise administering atherapeutic agent, e.g., an NTRK2 inhibitor (such as, e.g., apan-NTRK-1,2,3 inhibitor, or an NTRK2-specific inhibitor) or an NTRK2fusion inhibitor, i.e., an inhibitor that blocks the activity of theNTRK2 fusion but not wild type NTRK2 (such as, e.g., an antibodyspecific to a TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 fusion protein,or any one of the antibodies described above; or an RNA inhibitor thatrecognizes NTRK2 or the fusion junction of an NTRK2 gene fusion,including but not limited to siRNA, dsRNA, shRNA, or any other antisensenucleic acid inhibitor), alone or in combination with e.g., otherchemotherapeutic agents or procedures, in an amount sufficient to treata condition mediated by aberrant NTRK2 expression or activity, oroverexpression of NTRK2, by one or more of the following: e.g., impedinggrowth of a cancer, causing a cancer to shrink by weight or volume,extending the expected survival time of the patient, inhibiting tumorgrowth, reducing tumor mass, reducing size or number of metastaticlesions, inhibiting the development of new metastatic lesions,prolonging survival, prolonging progression-free survival, prolongingtime to progression, and/or enhancing quality of life.

In certain embodiments, the NTRK2 fusion proteins of the invention maybe inhibited by a NTRK2 inhibitor or a NTRK2 fusion inhibitor. In someembodiments, the therapeutic agent is a NTRK2 inhibitor, such as, e.g.,a compound, biological or chemical, which inhibits, directly orindirectly, the expression and/or activity of NTRK2. For example, theNTRK2 inhibitors may be an antibody (such as, e.g., antibodies specificto NTRK2) or a small molecule inhibitor. In some embodiments, theinhibitors may act directly on NTRK2 itself, modify the activity ofNTRK2, or inhibit the expression of NTRK2. In other embodiments, theinhibitors may indirectly inhibit NTRK2 activity by inhibiting theactivity of proteins or molecules other than NTRK2 itself. For example,the inhibitors may modulate the activity of regulatory kinases thatphosphorylate or dephosphoryiate NTRK2, interfere with binding ofligands, or inhibit the activity of interacting or downstream proteinsor molecules.

Exemplary small molecule inhibitors include pan-kinase inhibitors withactivity against several different kinases (including NTRK2) or specificinhibitors (e.g., inhibitors specific to NTRKs, or specific to NTRK2).Exemplary pan-kinase inhibitors include, but are not limited to thoselisted in International Patent Publications WO 2006/123113,WO2011/133637, WO 2012/116217, WO 2012/034091, and WO 2012/034095, WO2013/074518, and WO 2013/174876. Exemplary NTRK-specific inhibitorsinclude those described in WO 2011/006074. Each of these applications isincorporated herein in its entirety for its disclosure of suchinhibitors.

In some embodiments, the NTRK2 fusion protein is inhibited by an NTRK2fusion inhibitor, such as, e.g., an antibody that recognizes all or partof an NTRK2 fusion (such as, e.g., a TRIM24:NTRK2 fusion protein, anAFAP1:NTRK2 fusion protein, or a PAN3:NTRK2 fusion protein) but does notrecognize wild type NTRK2 or wild type fusion partner (such as, e.g.,TRIM24, AFAP1, or PAN3). In some embodiments, the NTRK2 fusion protein(such as, e.g., a TRIM24:NTRK2 fusion protein, an AFAP1:NTRK2 fusionprotein, or a PAN3:NTRK2 fusion protein) is inhibited by an agent thatinhibits transcription or translation of the fusion protein, e.g., anRNA inhibitor that recognizes the NTRK2 coding sequence, the bindingpartner (e.g., TRIM24, AFAP1, or PAN3), or the binding partner: NTRK2fusion junction, including but not limited to small interfering RNA(siRNA), double stranded RNA (dsRNA), short-hairpin RNA (shRNA), or anyother antisense nucleic acid inhibitor. In some embodiments, the NTRK2fusion inhibited is selected from all or a portion of any one of SEQ IDNOs: 1-8.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of a conditionmediated by aberrant NTRK2 expression or activity, or overexpression ofNTRK2, such as delaying or minimizing one or more symptoms associatedwith a cancer or a tumor harboring an NTRK2 fusion (such as, e.g.,TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2, as disclosed herein). Atherapeutically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other therapeuticagents, which provides a therapeutic benefit in the treatment ormanagement of the cancer. The term “therapeutically effective amount”can encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of the condition mediated by aberrant NTRK2expression or activity or overexpression of NTRK2, or enhances thetherapeutic efficacy of another therapeutic agent.

In certain embodiments, the cancer or tumor harboring an NTRK2 fusion islung cancer, such as, e.g., lung adenocarcinoma. In other embodiments,the cancer or tumor harboring an NTRK2 fusion is glioma, such as, e.g.,a low grade glioma. In some embodiments, the cancer or tumor harboringan NTRK2 fusion is squamous cell carcinoma, such as, e.g., head and necksquamous cell carcinoma.

In some embodiments, the patient to be treated is suffering from lungcancer, such as, e.g., lung adenocarcinoma, and the method for treatingthe condition comprises administering to the patient a therapeuticallyeffective amount of an NTRK2 inhibitor or an NTRK2 fusion inhibitor. Insome embodiments, the patient to be treated is suffering from glioma,such as, e.g., a lower grade glioma, and the method for treating thecondition comprises administering to the patient a therapeuticallyeffective amount of an NTRK2 inhibitor or an NTRK2 fusion inhibitor. Insome embodiments, the patient to be treated is suffering from squamouscell carcinoma, such as, e.g., head and neck squamous cell carcinoma,and the method for treating the condition comprises administering to thepatient a therapeutically effective amount of an NTRK2 inhibitor or anNTRK2 fusion inhibitor.

Screening Methods

Therapeutic agents, such as e.g., NTRK2 inhibitors or NTRK2 fusioninhibitors, used in the therapeutic methods of the invention can beevaluated using the screening assays described herein. Thus, theinvention provides a method of identifying an agent useful for treatinga condition mediated by aberrant NTRK2 expression or activity, oroverexpression of NTRK2, such as, e.g., a cancer or a tumor harboring anNTRK2 fusion, such as e.g., lung cancer (e.g., lung adenocarcinoma),glioma (e.g., low grade glioma), or squamous cell carcinoma (e.g., headand neck squamous cell carcinoma), comprising contacting a cellexpressing an NTRK2 gene fusion or NTRK2 fusion protein with a candidateagent and determining whether the expression level of the fusion isdecreased or a biological function associated with the fusion isaltered. In one embodiment, therapeutic agents can be evaluated in acell-free system, e.g., a cell lysate or in a reconstituted system. Inother embodiments, the therapeutic agents are evaluated in a cell inculture, e.g., a cell expressing an NTRK2 fusion (e.g., a mammaliancell, a tumor cell or cell line, a recombinant cell). In yet otherembodiments, the therapeutic agents are evaluated in vivo (e.g., anNTRK2 fusion-expressing cell present in a subject, e.g., an animalsubject (e.g., an in vivo animal model)).

Exemplary parameters to evaluate in determining the efficacy of atherapeutic agent for treating a condition mediated by aberrant NTRK2expression or activity, or overexpression of NTRK2, such as, e.g., acancer or a tumor harboring an NTRK2 fusion include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to an NTRK fusion protein; or a binding competition between aknown ligand and the candidate agent to an NTRK2 fusion protein;

(ii) a change in kinase activity, e.g., phosphorylation levels of anNTRK fusion protein (e.g., an increased or decreased phosphorylation orautophosphorylation); or a change in phosphorylation of a target of anNTRK2 kinase—in certain embodiments, a change in kinase activity, e.g.,phosphorylation, is detected by any of western blot (e.g., using ananti-NTRK2 antibody or a phosphor-specific antibody, detecting a shiftin the molecular weight of an NTRK2 fusion protein), mass spectrometry,immunoprecipitation, immunohistochemistry, immunomagnetic beads, amongothers;

(iii) a change in an activity of a cell containing an NTRK fusion (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology, or tumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, or proliferation of the tumor; or

(v) a change in the level, e.g., expression (transcription and/ortranslation) level of an NTRK2 fusion protein or nucleic acid molecule;or

(vi) a change in an activity of a signaling pathway involving NTRK2,e.g., phosphorylation or activity of an interacting or downstreamtarget, or expression level of a target gene. In some embodiments, theNTRK fusion is a TRIM24:NTRK2 fusion, an AFAP1:NTRK2 fusion, or aPAN3:NTRK2 fusion.

In one embodiment, a change in the activity of an NTRK2 fusion, orinteraction of an NTRK2 fusion with a downstream ligand detected in acell free assay in the presence of a candidate agent indicates that thecandidate agent will be effective as a therapeutic agent for treatmentof a condition mediated by aberrant NTRK2 expression or activity, oroverexpression of NTRK2, such as, e.g., a cancer or a tumor harboring anNTRK2 fusion (such as, e.g., lung cancer (e.g., lung adenocarcinoma),glioma (e.g., low grade glioma), or squamous cell carcinoma (e.g., headand neck squamous cell carcinoma)).

In other embodiments, a change in an activity of a cell expressing anNTRK2 fusion, such as, e.g., TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2,as disclosed herein (e.g., a mammalian cell, a tumor cell or cell line,a recombinant cell) is detected in a cell in culture. In one embodiment,the cell is a recombinant cell that is modified to express an NTRK2fusion nucleic acid, e.g., is a recombinant cell transfected with anNTRK2 fusion nucleic acid. The transfected cell can show a change inresponse to the expressed NTRK2 fusion, e.g., increased proliferation,changes in morphology, increased tumorigenicity, and/or acquired atransformed phenotype. A change in any of the activities of the cell,e.g., the recombinant cell, in the presence of the candidate agent canbe detected. For example, a decrease in one or more of: proliferation,tumorigenicity, or transformed morphology, in the presence of thecandidate agent can be indicative of an inhibitor of an NTRK2 fusion. Inother embodiments, a change in binding activity or phosphorylation ofNTRK2 or its interacting or downstream proteins or molecules asdescribed herein is detected.

In yet other embodiment, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,a tumor containing animal or a xenograft comprising cells expressing anNTRK fusion (e.g., tumorigenic cells expressing an NTRK fusion) isemployed. The therapeutic agents can be administered to the animalsubject and a change in the tumor is evaluated. In one embodiment, thechange in the tumor includes one or more of a tumor growth, tumor size,tumor burden, or survival, is evaluated. A decrease in one or more oftumor growth, tumor size, tumor burden, or an increased survival isindicative that the candidate agent is an inhibitor or modulator.

In another aspect of the invention provides a method or assay forscreening for agents that modulate (e.g., inhibit) the expression oractivity of an NTRK2 fusion as described herein. The method includescontacting e.g., an NTRK2 fusion, or a cell expressing an NTRK2 fusion,with a candidate agent; and detecting a change in a parameter associatedwith an NTRK2 fusion, e.g., a change in the expression or an activity ofthe NTRK2 fusion. The method can, optionally, include comparing thetreated parameter to a reference value, e.g., a control sample (e.g.,comparing a parameter obtained from a sample with the candidate agent toa parameter obtained from a sample without the candidate agent). In oneembodiment, if a decrease in expression or activity of the NTRK2 fusionis detected, the candidate agent is identified as an inhibitor. Inanother embodiment, if an increase in expression or activity of theNTRK2 fusion is detected, the candidate agent is identified as anactivator. In certain embodiments, the NTRK fusion is an NTRK2 genefusion or NTRK2 fusion protein, where in the fusion is e.g., aTRIM24:NTRK2 fusion, an AFAP1:NTRK2 fusion, or a PAN3:NTRK2 fusion.

In one embodiment, the contacting step is detected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is detected in a cell in culture, e.g.,a cell expressing an NTRK2 fusion (e.g., a mammalian cell, a tumor cellor cell line, a recombinant cell). In yet other embodiments, thecontacting step is detected in vivo (e.g., an NTRK2 expressing cellpresent in a subject, e.g., an animal subject (e.g., an in vivo animalmodel)).

Exemplary parameters evaluated in identifying an agent that modulatesthe activity of an NTRK fusion, e.g., an NTRK2 fusion (e.g., aTRIM24:NTRK2 fusion, an AFAP1:NTRK2 fusion, or a PAN3:NTRK2 fusion)include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to an NTRK2 fusion protein; a binding competition between a knownligand and the candidate agent to an NTRK fusion protein;

(ii) a change in kinase activity, e.g., phosphorylation levels of anNTRK2 fusion protein (e.g., an increased or decreased phosphorylation orautophosphorylation); or a change in phosphorylation of a target of anMIRK kinase—in certain embodiments, a change in kinase activity, e.g.,phosphorylation, is detected by any of western blot (e.g., using ananti-NTRK2 antibody or a phosphor-specific antibody, detecting a shiftin the molecular weight of an NTRK2 fusion protein), mass spectrometry,immunoprecipitation, immunohistochemistry, immunomagnetic beads, amongothers;

(iii) a change in an activity of a cell containing an NTRK fusion (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology, or tumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, or proliferation of the tumor;

(v) a change in the level, e.g., expression (transcription and/ortranslation) level of an NTRK2 fusion protein or nucleic acid molecule;or

(vi) a change in an activity of a signaling pathway involving NTRK2,e.g., phosphorylation or activity of an interacting or downstreamtarget, or expression level of a target gene.

Methods for Validating NTRK Fusions

NTRK gene fusions (such as, e.g., TRIM24:NTRK2 gene fusions, AFAP1:NTRK2gene fusions, or PAN3:NTRK2 gene fusions) may be evaluated to ensurethat the breakpoints are in-frame and can produce a protein productcontaining the full kinase domain, i.e., that the breakpoint occurs suchthat complete triplet codons are intact, and that the RNA sequence willproduce a viable protein. The NTRK gene fusion can be transfected intocells to confirm that the protein is functionally active with respect tokinase activity and oncogenic activity. cDNA encoding the NTRK fusionprotein can be produced by standard solid-phase DNA synthesis.Alternatively the NTRK fusion cDNA can be produced by RT-PCR using tumormRNA extracted from samples containing the gene fusion. The DNAsubcloned can be cloned into an appropriate vector and characterized byDNA sequence analysis or in vitro/in vivo expression analyses.

Expression vectors containing the NTRK gene fusion (such as, e.g., aTRIM24:NTRK2 gene fusion, an AFAP1:NTRK2 gene fusion, or a PAN3:NTRK2gene fusion) can be introduced into host cells to thereby produce anNTRK2 fusion protein (such as, e.g., a TRIM24:NTRK2 fusion protein, anAFAP1:NTRK2 fusion protein, or a PAN3:NTRK2 fusion protein). The NTRK2fusion protein expression vector can be a yeast expression vector, avector for expression in insect cells, e.g., a baculovirus expressionvector, or a vector suitable for expression in mammalian cells. VectorDNA can be introduced into host cells via conventional transformation ortransfection techniques. As used herein, the terms “transformation” and“transfection” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell.

Cells harboring the expression vector carrying the recombinant NTRK genefusion can then be tested for production of the unique fusion proteinvia standard western blotting using either an antibody probe thatdetects the gene product itself or that recognizes a tag peptide (e.g.,FLAG tag) that can be added to the gene product via the expressionvector (using standard, commercially available reagents). Westernblotting can be used to confirm the ectopic expression of the encodedNTRK2 fusion protein by comparing the samples from cells transfectedwith the vector containing the NTRK2 gene fusion cDNA to cellstransfected with the empty expression vector. The functional activitycan be assessed by measuring the level of phosphorylation on the kinaseor substrate. Comparison of the level of phosphorylation activitybetween the wild type (normal) form of NTRK2 and the NTRK2 fusionprotein can indicate if the NTRK2 fusion protein has elevated activitythat could drive oncogenic activity. Whether the NTRK gene fusion isoncogenic can be assessed by measuring capacity of the expressed NTRK2fusion protein to transform cells, that is, to enable cells to grow andproliferate under conditions which are not permissive for growth ofnormal cells. One commonly used method of measuring the transformingactivity of a kinase is by assessing if expression of the gene productcan allow BaF3 cells to grow in the absence of the growth factor IL3,which is required for the survival and growth of BaF3 cells. Anotherassay for measuring transforming activity is a soft agar growth assay.This is another standard method which tests the capacity of anintroduced gene product to confer the ability to grow in a soft agarmatrix, or anchorage-independent conditions. These methods and otherscan be used to test the oncogenic activity of an NTRK2 gene fusion (suchas, e.g., a TRIM24:NTRK2 gene fusion, an AFAP1:NTRK2 gene fusion, aPAN3:NTRK2 gene fusion) and provide a level of validation of an NTRK2fusion protein (such as, e.g., a TRIM24:NTRK2 fusion protein, anAFAP1:NTRK2 fusion protein, or a PAN3:NTRK2 fusion protein) as apotential target for treating patients that harbor these fusions.

A change in an activity of a cell can be detected in a cell in culture,e.g., a cell expressing a fusion (e.g., a mammalian cell, a tumor cellor cell line, a recombinant cell). The transfected cell can show achange in response to the expressed fusion, e.g., increasedproliferation, changes in morphology, increased tumorigenicity, and/oran acquired transformed phenotype.

To further validate the biological implication of the gene fusion, achange in any of the activities of the cell, e.g., the recombinant cell,in the presence of a known inhibitor of one of the fusion partners,e.g., an NTRK2 inhibitor, can be detected. For example, a decrease inone or more of: proliferation, tumorigenicity, or transformedmorphology, in the presence of the NTRK2 inhibitor can be indicative ofan inhibitor of a fusion. In other embodiments, a change in bindingactivity or phosphorylation of NTRK2 or its interacting or downstreamproteins or molecules as described herein is detected.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification will supersede any contradictorymaterial. Unless otherwise required by context, singular terms shallinclude the plural and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. All ranges given in the application encompass theendpoints unless stated otherwise.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-81. (canceled)
 82. A compound capable of specifically inhibiting aTRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 fusion, wherein the fusionresults in aberrant activity or expression of NTRK2, or overexpressionNTRK2, and wherein the compound is an antibody that specifically bindsto a TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 fusion protein or an RNAinhibitor that hybridizes under stringent conditions to a TRIM24:NTRK2,AFAP1:NTRK2, or PAN3:NTRK2 gene fusion.
 83. The compound of claim 82,wherein (a) the antibody specifically binds to: (i) a fragment of SEQ IDNO:3 comprising at least amino acids 665-674 of SEQ ID NO:3; (ii) afragment of SEQ ID NO:4 comprising at least amino acids 665-674 of SEQID NO:4; (iii) a fragment of SEQ ID NO:6 comprising at least amino acids544-553 of SEQ ID NO:6; or (iv) a fragment of SEQ ID NO:8 comprising atleast amino acids 138-147 of SEQ ID NO:8; and (b) the RNA inhibitorhybridizes under stringent conditions to: (i) a fragment of SEQ ID NO:1comprising at least nucleotides 2010-2019 of SEQ ID NO:1; (ii) afragment of SEQ ID NO:2 comprising at least nucleotides 2000-2009 of SEQID NO:2; (iii) a fragment of SEQ ID NO:5 comprising at least nucleotides1641-1650 of SEQ ID NO:5; or (iv) a fragment of SEQ ID NO:7 comprisingat least nucleotides 426-435 of SEQ ID NO:7.
 84. A method for detectingthe presence of an NTRK2 fusion selected from an AFAP1:NTRK2 genefusion, a PAN3:NTRK2 gene fusion, a TRIM24:NTRK2 protein fusion, anAFAP1:NTRK2 protein fusion, and a PAN3:NTRK2 protein fusion in apatient, said method comprising: a) contacting a biological sample fromthe patient with a reagent that specifically binds to the fusionjunction of the NTRK2 fusion, and detecting binding between the NTRK2fusion and the reagent; or b) amplifying or sequencing a portion of anucleic acid from the patient, and detecting the presence of anucleotide sequence comprising at least the fusion junction of the NTRK2fusion.
 85. The method of claim 84, wherein the NTRK2 fusion to bedetected is an NTRK2 gene fusion that comprises SEQ ID NO:5 or 7, or aportion of SEQ ID NO:5 or 7, wherein the portion encodes a polypeptidehaving NTRK2 kinase activity and comprises the fusion junction.
 86. Themethod of claim 85, wherein the reagent is an oligonucleotide thathybridizes to the fusion junction of the NTRK2 gene fusion understringent conditions.
 87. The method of claim 86, wherein theoligonucleotide hybridizes under stringent conditions to (a) a fragmentof SEQ ID NO:5 comprising at least nucleotides 1641-1650 of SEQ ID NO:5;or (b) a fragment of SEQ ID NO:7 comprising at least nucleotides 426-435of SEQ ID NO:7.
 88. The method of claim 84, wherein the NTRK2 fusion tobe detected is an NTRK2 fusion protein that comprises SEQ ID NO:3, 4, 6,or 8, or a portion of SEQ ID NO:3, 4, 6, or 8, wherein the portion hasNTRK2 kinase activity and comprises the fusion junction.
 89. The methodof claim 88, wherein the reagent is an antibody that specifically bindsto the NTRK2 fusion protein, but does not specifically bind to wild-typeNTRK2 or wild-type TRIM24, AFAP1, or PAN3.
 90. The method of claim 89,wherein the antibody specifically binds to (a) a fragment of SEQ ID NO:3comprising at least amino acids 665-674 of SEQ ID NO:3; (b) a fragmentof SEQ ID NO:4 comprising at least amino acids 665-674 of SEQ ID NO:4;(c) a fragment of SEQ ID NO:6 comprising at least amino acids 544-553 ofSEQ ID NO:6; or (d) a fragment of SEQ ID NO:8 comprising at least aminoacids 138-147 of SEQ ID NO:8.
 91. The method of claim 84, furthercomprising administering to the patient a therapeutically effectiveamount of an NTRK2 inhibitor or an NTRK2 fusion inhibitor.
 92. Themethod of claim 84, wherein the patient is suffering from or susceptibleto a cancer.
 93. The method of claim 92, wherein the cancer is lungadenocarcinoma, low grade glioma, squamous cell carcinoma, or head andneck squamous cell carcinoma